Characteristic impairment of progesterone response in cultured cervical fibroblasts obtained from patients with refractory cervical insufficiency

Preterm birth (PTB) is the leading cause of neonatal mortality, and reducing the PTB rate is one of the most critical issues in perinatal medicine. Cervical insufficiency (CI), a major cause of PTB, is characterised by premature cervical ripening in the second trimester, followed by recurrent pregnancy loss. Although multiple clinical trials have suggested that progesterone inhibits cervical ripening, no studies have focused on progesterone-induced molecular signalling in CI. Here, we established a primary culture system for human uterine cervical fibroblasts using a sample of patients with refractory innate CI who underwent transabdominal cervical cerclage and patients with low Bishop scores who underwent elective caesarean section as controls. RNA sequencing showed that the progesterone response observed in the control group was impaired in the CI group. This was consistent with the finding that progesterone receptor expression was markedly downregulated in CI. Furthermore, the inhibitory effect of progesterone on lipopolysaccharide-induced inflammatory stimuli was also impaired in CI. These results suggest that abnormal cervical ripening in CI is caused by the downregulation of progesterone signalling at the receptor level, and provide a novel insight into the molecular mechanism of PTB.


Results
Cervical tissue sampling. Cervical tissue samples were collected from pregnant women with refractory cervical insufficiency (CI) characterized by a matured cervix (n = 3) and from pregnant women without cervical maturity as controls (n = 3). CI was defined as a pregnancy history of miscarriage preceded by asymptomatic cervical dilation and prolapse of membrane in the second trimester. The current pregnancies of the patients were maintained until 35 weeks of gestation with TAC. Controls included patients with no history of mid-term miscarriages or PTB. They had undergone an elective caesarean section due to their pelvic position or previous caesarean section before labour with an immature cervix (Bishop score < 4 points). After conization, patients with bacterial vaginitis, clinical chorioamnionitis, hypertensive disorder, and/or gestational diabetes mellitus were excluded from the study. Bacterial vaginitis was diagnosed on the basis of the Nugent score. A breakdown of these cases is shown in Table 1.
Comprehensive analysis of gene expression response to P4 addition. To investigate the gene expression change induced by P4, we performed whole-transcriptome analysis using RNA sequencing (RNAseq). Uterine cervical fibroblasts (UCFs) derived from the control (n = 2) and CI (n = 2) were precultured with 10 nmol/L beta-oestradiol in duplicate for 24 h and subsequently incubated with progesterone (1 μM) or ethanol alcohol (solvent) alone for 6 h. RNA was extracted from each sample and subjected to RNA-seq analysis. Gene set enrichment analysis (GSEA) of Molecular Signatures Database (MSigDB) hallmark gene sets (Table 2) showed no trend leading to pathophysiology when comparing control and CI under conditions without P4. In control-derived UCFs, P4 administration enhanced the expression of the gene set associated with sex hor- www.nature.com/scientificreports/ mone response and suppressed the proinflammatory gene set, including TNFα signalling via NFκB, inflammatory response, and interferon response. In contrast, in CI-derived UCFs, P4 administration did not change the expression of the gene set associated with sex hormone response but enhanced the expression of the proinflammatory gene set, including TNFα signalling and interferon response. Additionally, P4 administration produced no significant trend in the extracted gene sets. Next, a heat map was created based on the expression levels of 387 genes whose expressions were changed by P4 administration (p < 0.05). The entire list of 387genes, consisting of 150 upregulated and 237 downregulated genes, can be found in Supplementary Tables S1 and S2. Both up-and downregulated control-derived UCFs showed a common trend of expression change in response to P4, whereas no response to P4 was observed in CI-derived UCFs (Fig. 1).
Comparative analysis of P4 response focusing on specific molecules. The RNA sequencing analyses demonstrated that contrary to our initial expectation, the molecular groups reported to be involved in prior cervical ripening, including Has2 26, 27 , VCAN, THBS1, COL5A1, and MYOCD 28 , did not show significant gene expression changes between cells obtained from control and CI patients in response to P4 stimulation. This finding was also confirmed by the real-time PCR analyses ( Supplementary Fig. 2).
Among the upregulated genes as determined by RNA-seq analysis, FKBP5 (FKBP Prolyl Isomerase 5) 29, 30 and SPARCL1 (SPARC-Like Protein 1) 31, 32 are representative P4-responsive genes aligned with previous reports. We designed primers for FKBP5, a glucocorticoid receptor co-chaperone, and SPARCL1, a secreted glycoprotein that constitutes the extracellular matrix. P4 (1 μM) dissolved in ethanol or ethanol as a control was added to the UCFs culture system, RNA was extracted 6 h later, and an inverted-copy reverse transcriptase RT-PCR was used to analyse the complementary cDNA. FKBP5 and SPARCL1 expression level was significantly upregulated in controls following P4 treatment (p < 0.05) while it remained unaffected in the CI-derived cells (Fig. 2).
Analysis of progesterone receptor expression. Next, we focused on the differences in progesterone receptor (PR) expression levels in fibroblasts as a possible mechanism for the differential P4 response. According Table 2. Comparison of gene expression by Hallmarks Gene Set Enrichment Analysis. Gene sets differentially expressed between control and CI in no treatment and up or downregulated gene sets by P4 in control, and CI are shown. Statistical significance was set at p < 0.05, and the top eight gene sets are shown. www.nature.com/scientificreports/ to the results of RNA sequence analysis, P4 addition did not change the PR expression level in either CI-derived or control-derived UCFs. Cell immunostaining also showed no difference in PR expression due to the addition of P4 (data not shown). Therefore, a comparative analysis of PR expression was performed by real-time PCR, western blotting, and cell immunostaining, using CI-derived and control-derived UCFs treated with P4. After 24 h of incubation with E2, cells were harvested for comparative analysis of the expression of the two isoforms of PR, PR-A and PR-B. Real-time PCR revealed no significant differences in the gene expression of the progesterone receptor gene (PGR); however, a significant (p < 0.05) decrease in the expression of PGR-B, an isoform with a more extended transcription region, was observed in the CI group compared to that in the control group (Fig. 3A). Western blotting analysis of protein expression levels also showed no bands corresponding to PR-A and PR-B in CI (Fig. 3B). The photograph of the full-length gel is shown in Supplementary Fig. S1. In immunocytochemistry, the PR was stained in the nuclei of the control group and not in the nuclei of the CI group (Fig. 3C). The comparably weak cytoplasmic staining in both CI-derived and control-derived UCFs can be regarded as a non-specific reaction, based on the western blot analysis showing non-specific bands in both UCFs conducted using the same antibody.

Analysis of inflammatory response to LPS.
To investigate the effect of differential P4 signalling on the inflammatory response, next used real-time PCR to quantitatively analyse the inhibitory effect of P4 (1 µM) on the inflammatory response to LPS, which are exogenous ligands. UCFs were treated with LPS (2 µg/mL) for 1 h,  www.nature.com/scientificreports/ then P4 (1 μM) dissolved in ethanol or ethanol as a control was added for 6 h, RNA was extracted, and cDNA was reverse-transcribed and analysed using RT-PCR. The suppressive effect of P4 on the induction of IL-1B, IL-6, and prostaglandin-endoperoxide synthase 2 (PTGS2) expression by LPS was observed in the control group but not in the CI group (Fig. 4). This suggests that the P4 signalling-mediated suppressive effect on the inflammatory response to LPS is impaired in the cervical tissues of CI.

Discussion
In this study, the response to P4 administration observed in cultured UCFs derived from pregnant women with an unripened cervix was completely diminished in those UCFs derived from patients with refractory CI. We further confirmed that a pronounced decrease in the expression of progesterone receptors accompanied this loss of response to P4 administration. This explains the putative molecular mechanism underlying refractory  www.nature.com/scientificreports/ endogenous CI, which is also consistent with the concept that human cervical ripening is caused by the withdrawal of P4 signalling. Obtaining fully ripened cervical tissue unaffected by labour and/or delivery is difficult, and the existing study used cervical tissue obtained immediately after parturition. With this study design, it is impossible to assess the molecular change accompanied by cervical ripening itself, because mechanical stress to the cervix due to cervical dilatation and the systemic maternal change associated with labour pains may affect the molecular expression. In the present study, the unique clinical sample from a ripened cervix of patients who underwent TAC enabled us for the first time to investigate pure molecular changes associated with cervical ripening while excluding the effects of delivery for the first time.
P4 is essential for maintaining pregnancy, and the administration of PR antagonists is known to induce rapid cervical ripening. There are two isoforms of PR, PR-A and PR-B. The PR-B signal primarily contributes to the maintenance of pregnancy, and its function is regulated by the expression balance of PR-A [33][34][35] . In this study, UCFs cultures derived from patients with innate refractory CI showed a marked decrease in PR expression at the protein level and a more pronounced downregulation of the functional receptor PR-B at the mRNA level. As indicated in the heatmap, the overall progesterone response in CI was significantly attenuated. However, as shown in Table 2, partial responses were observed in CI. It has been reported that progesterone can also signal through the glucocorticoid receptor (GR). We found no significant difference in GR expression between the control and CI (Supplementary Fig. 3). Therefore, while the response mediated by PR was diminished, we speculate that residual partial progesterone response may be attributable to crosstalk mediated by GR. In a control UCFs culture system established from the immature cervix of full-term pregnant women, genes responsive to P4 administration were found to be similar to those in a previous comprehensive expression analysis report using non-pregnancy cervical tissue 36 .
According to GSEA, the functional roles of these genes are involved in the suppression of inflammatory cytokines, which is consistent with the results of previous studies that focused on P4 signalling in cervical tissue 24 . As these genes are considered responsible for the maintenance of pregnancy via PR signalling, they might be important for elucidating the mechanisms of cervical ripening and constitute candidates for new therapeutic targets and/or biomarkers for predicting the risk of PTB.
In this study, we found that cases of refractory CI showed acute cervical ripening in the second trimester. We further analysed the UCFs established from the cervical stroma and found that those from patients with refractory innate CI lost a major part of PR expression and were characterized by an impaired P4 response. These findings strongly suggest that an impaired P4 response caused by decreased PR expression is involved in some refractory cases of endogenous CI. We assume that in such cases, the expression of PR in cervical stromal cells will be suppressed during mid-gestation, resulting in rapid maturation and leading to exposure and rupture of the foetal membranes.
At present, it is unclear what causes such differences in PR expression levels. ER signalling is known as an upstream factor that promotes PR expression. All experiments in this study were performed using UCF precultured for 24 h in media containing beta-oestradiol in the expectance of inducing stable PR expression. According to the real-time PCR analyses, no significant differences in ER expression were observed between control and CI cells (Supplementary Fig. 3). Considering that large amounts of oestrogen are constantly produced by the placenta during pregnancy, it is difficult to explain differences in PR expression levels in relation to ER signalling.
As this unique property was maintained even after several subcultures, it is reasonable to assume that it is based on inherited genetic or epigenetic factors. As there were no problems with the establishment of pregnancy or its course until the first trimester, congenital PR deficiency may not be involved. It appears that these phenomena are caused by a specific unknown mechanism during the second trimester of pregnancy when premature cervical ripening becomes apparent. Exploring this mechanism will provide data for understanding how the trigger of P4 withdrawal that precedes parturition is regulated in both pre-and full term instances. As a next step, we plan to conduct an integrated omics analysis using whole genome sequence data from patients with endogenous CI and epigenome data (by ChIP-or ATAC-sequence) from each sample, in addition to elucidating the RNA sequence data.
From a clinical perspective, preventive P4 therapy, which is widely used for pregnant women at high risk of premature birth, is considered partially effective or ineffective for patients with refractory innate CI. There are currently no studies investigating the therapeutic effects of progesterone focused on refractory CI patients, which constitutes an important topic for future investigations. Also, further investigation is needed to determine whether this putative pathophysiological mechanism characterized by loss of PR expression can be applied to cervical ripening observed prior to other types of PTB and/or term delivery.
The study was subject to some limitations. First, we used a small number of cases, as we targeted the rare instances of patients with refractory endogenous CI who underwent TAC. Second, because of the nature of the experiment using the primary culture system, the number of samples that could be used for each experiment was limited to avoid property changes due to passage. Increasing the number of cases to verify the generalizability and validity of the study results is challenging due to the inclusion of the invasive cervical tissue biopsy procedure. In the future, the use of non-invasive methods such as endocervical scraping samples and/or cervical mucus collected during pregnancy could be valuable for indirectly assessing changes in PR expression. By analysing the P4-responsive molecules identified in these samples, it would be possible to evaluate PR expression.
In conclusion, the impaired P4 response accompanied by decreased PR expression was characteristic of cultured cervical fibroblasts obtained from patients with refractory CI. Further investigations targeting sensitive P4-responsive molecules and elucidating the regulatory mechanisms of PR expression are essential for the development of new clinical strategies for the prevention of PTB.

Methods
Establishment of primary UCFs culture. As previously reported, we prepared UCFs from uterine tissues 23 . All samples were collected with written informed consent obtained from the subjects. After delivery of the foetus and placenta in a scheduled caesarean section before the onset of labour, we identified the closed endocervix through the uterine wound and collected 5 mm of cervical tissue. The study protocol was approved by the Nippon Medical School Hospital Ethics Committee (approval number: R1-08-1176) and was carried out in accordance with the Ethical Guidelines for Medical and Biological Research Involving Human Subjects. The uterine cervical tissue was cut into small pieces and incubated in Dulbecco's modified Eagle's medium (Gibco/Invitrogen, Carlsbad, CA, USA) containing 10% foetal bovine serum (HyClone, Logan, UT, USA) in a humidified atmosphere of 5% CO 2 and 95% air at 37 °C. The culture medium was changed twice per week. Once 80% confluent, fibroblasts were enzymatically detached with 0.25% trypsin (Gibco) and cultured in 10 cm culture plates. All experiments were performed using cells from passages 4 to 8. UCFs were precultured for 24 h in media containing 10 nmol/L beta-oestradiol conforming to the physiological conditions present during pregnancy and subsequently incubated in the presence or absence of P4 and LPS isolated from Escherichia coli (L2880, Sigma-Aldrich, St. Louis, MO, USA).  . Amplification and detection were performed using the StepOnePlus system (Applied Biosystems). The amplification conditions consisted of pre-incubation at 95 °C for 20 s, 40 cycles of denaturation at 95 °C for 3 s, and annealing at 60 °C for 30 s, followed by a melting curve analysis to confirm that the target amplicons were detected. The fluorescence data were analysed using StepOne Software. The samples were assayed in triplicate for each gene and the mean expression was used for subsequent analyses. GAPDH expression was used to normalise the mRNA levels in each sample. Relative expression was calculated using the ∆∆Ct method.

RNA sequencing.
Immunocytochemistry. To  www.nature.com/scientificreports/ Western blotting analysis. To analyse PR expression, the cells were incubated in the presence of 10 nM/L β-oestradiol for 24 h. Cell lysate (10 μg) was separated on a 4-20% Mini-PROTEAN TGX Gel (BioRad, Hercules, CA, USA) and electrophoretically transferred to a polyvinylidene fluoride (PVDF) membrane (Trans-Blot Turbo Mini PVDF Transfer Packs; BioRad). The membrane was cut to an appropriate size prior to the following experimental procedures. First, the membranes were blocked in PVDF Blocking Reagent for Can Get Signal (Toyobo) and subsequently incubated with primary antibody in Can Get Signal Solution 1 for 12 h at 4 °C. Thereafter, membranes were washed with Tris buffer (20 mM/L Tris-HCl, pH 7.6; 137 mM/L NaCl; 0.1% Tween-20) and incubated with a secondary antibody in Can Get Signal Solution 2. For detection of PR, the primary antibody was rabbit anti-Progesterone Receptor A/B (D8Q2J; 1:5000 dilution), and the secondary antibody was ECL anti-rabbit IgG Horseradish Peroxidase-linked antibody (NA934, GE Healthcare, Chicago, IL, USA; 1:20,000 dilution). In addition, the anti-GAPDH antibody (5A12, Wako Co., Ltd., Tokyo, Japan; 1:50,000 dilution) and ECL anti-mouse IgG Horseradish Peroxidase-linked antibody (NA931, GE Healthcare; 1:50,000 dilution) were used as internal controls. Proteins were subsequently visualized using ECL Prime Western Blotting Detection Reagents (GE Healthcare) and imaged using a LAS-4000 Luminescent Image Analyzer (FUJIFILM Co., Tokyo, Japan). Images of PR and GAPDH staining were obtained using the same lane of the same gel, with an exposure time of 300 s for PR and 60 s for GAPDH.

Data availability
All RNA-seq data are available in the GEO under accession number GSE220806. Currently, token "etqnqiegljohraz" is issued for access.